The present invention relates to the field of microbial detection in clinical samples. The invention is in particular related to achieving faster detection of the presence or absence of bacteria in a biological sample.
The detection of the presence or absence of microbes (e.g. bacteria) in a biological sample is a necessary aspect of health care. Typically such detection requires that the microbes be cultured to provide enough microbes to be detected. There is a broad array of culture media for the growth of microbes in a sample, as the presence or absence of the microbes in the sample can only be determined if the quantity of microbes in the test sample is sufficient to ensure that the microbes will be detected if they are present.
For example, bacteria in clinical blood samples are typically detected by inoculating approximately 10 ml of whole blood in a culture bottle containing approximately 30 mL of growth media to support bacterial multiplication. The sample incubates in the bottle in an automated system at 35° C. The sample is monitored for the byproducts of cell metabolism or cell growth to determine the presence or absence of bacteria in the sample. In one example, the products of bacterial metabolism (such as carbon dioxide) are monitored by means of chemical sensors disposed within the culture bottle.
The presence of a growing bacterial population within a culture bottle of 80 mL overall volume is typically detected when the number of microorganisms has risen to approximately 5×109 CFU (colony forming units). It is obvious that many bacterial doubling events are required to grow a bacterial population from one or two organisms in the 10 mL blood sample to such a high number. One solution to providing faster bacterial detection is splitting the 10-mL sample liquid together with the required growth media (typically 30 mL volume of growth media is combined with the 10 mL of blood) into a large number of smaller partial samples that are contained in closed small chambers. This is described in U.S. Pat. Nos. 5,770,440 and 5,891,739 to Berndt, which are incorporated by reference herein. U.S. Pat. No. 5,716,798 to Monthony et al., which is incorporated by reference herein, describes an array of small chambers (a 96 well array of 250 μl wells) that are not closed from each other, but have a joint head space volume. Monthony et al. contemplates the use of colorimetric, fluorometric, radiometric, nephelometric, and infrared analysis to assay the sample well to detect the presence or absence of bacteria therein. Monthony et al. reports that a shortening in the time to detection (TTD) is achieved with smaller sample volumes.
While the splitting of the original 10-mL blood sample together with the 30 mL of growth media is promising towards achieving faster bacterial detection, the design of a practical multi-chamber sample container for detecting the presence or absence of microorganisms in the one or more chambers is a challenge. For example, if bacterial growth is detected in only one or two of the small chambers, then these chambers need to be identified and accessed in order to remove the sample liquid from those chambers where positive growth is detected for downstream analysis such as ID (e.g. Maldi time-of-flight) and antibiotic susceptibility testing (AST). Accurately removing sample from discrete chambers in an array of small chambers represents a further challenge.
Another challenge to the implementation of an array of small-volume chambers for detecting microbial growth is the detectors that are deployed. Optical interrogation of the individual chambers requires accurate measurements to ensure that the measurement is associated with the appropriate chamber. Signal cross talk from well to well also must be avoided. The deployment of individual chemical sensors for each well can be expensive and difficult to implement.
Dielectric impedance measurement has been evaluated as an alternative to the use of chemical sensors. However, barriers to commercial deployment include the sensitivity of the impedance to temperature fluctuations. Maintaining the temperature of the blood culture bottle to better than +/−0.05° C. is not practical for a clinical bacterial detection environment.
In Sengupta, S, et al., “A micro-scale multi-frequency reactance measurement technique to detect bacterial growth at low bio-particle concentrations,” Lab Chip, Vol. 6, pp. 682-692 (2006), which is incorporated by reference herein, a micro-fluidic chamber of 100 μl volume was used as the chamber for sensing response to the presence of bacteria. Sengupta et al. reported that the sensing response can be improved relative to a simple dielectric conductivity measurement by providing a long and very thin channel-like chamber containing the sample, with very small electrodes positioned at both ends. By using high frequencies up to 100 MHz, the capacitive contribution of the liquid sample was measured, which, according to Sengupta et al., is more sensitive to the changes in capacitance in the sample caused by the presence and/or growth of bacteria in the chamber.
As further described in Sengupta, S., et al., “Rapid detection of bacterial proliferation in food samples using microchannel impedance measurements at multiple frequencies,” Scns. & Instrumen. Food Qual., Vol. 4, pp. 108-118 (2010) and Puttaswamy, S., et al., “Novel Electrical Method for Early Detection of Viable Bacteria in Blood Cultures,” J. Clin. MicroBio., Vol. 49(6), pp. 2286-2289 (2011), both of which are incorporated by reference herein, temperature fluctuations are described as the most significant challenge to the use of the Sengupta et al. apparatus and method of using a microfluidic environment to assay for the presence of bacteria in a sample using a dielectric conductivity measurement.
A further limit on the Sengupta et al. apparatus and method is the need to fill a new microfluidics chamber (or replace the liquid sample in the microfluidics chamber with fresh liquid sample from the culture bottle) after one hour or so and make the next measurement with a new sample. This approach consumes approximately 1 mL of sample liquid within ten hours, as each previously sampled portion is discarded. While sampling could happen more often to achieve a better signal-to-noise ratio; for slow growing microorganisms, the volume of sample consumption over time could represent a serious challenge.
Therefore, there exists the need for improvement if the use of dielectric measurements to detect the presence or absence of microbes in a liquid sample is to be commercially viable.